Current egg-based influenza vaccine manufacturing technology requires approximately six months to prepare sufficient doses of seasonal trivalent inactivated influenza vaccine (TIV). On several occasions in recent years, manufacturing issues have created vaccine shortages that resulted in a subsequent prioritization of vaccine recipients. Furthermore, limited surge capacity exists beyond the yearly 80-90 million doses of TIV produced for U.S. consumption, which will pose constraints on vaccine manufacture in the event of a pandemic outbreak. The current WHO Phase 3 pandemic alert status of avian to human H5N1 influenza virus infections and deaths has encouraged the development of new vaccine technologies capable of producing large numbers of doses under tightly compressed timelines. Because it is not possible to predict the exact virus strain prior to an influenza outbreak, rapid vaccine production capability is required for a timely response to an influenza pandemic. The ultimate aim of this research is to develop a manufacturing system capable of generating millions of bulk doses of hemagglutinin (HA)-based DNA vaccines within a very short time. Utilizing novel manufacturing technology already under development at Vical Incorporated, HA-based DNA vaccines would be produced against an emerging influenza strain within days to weeks after acquiring the emerging HA genomic sequence. In addition, this manufacturing system would be used during interpandemic periods to produce yearly seasonal trivalent vaccines without the constraints of the current egg-based procedures. The present aim of this proposal is to develop and characterize a polymerase chain reaction (PCR)-based manufacturing system that can rapidly produce vaccine doses comprising bulk, linear DNA expression cassettes (LECs) encoding an influenza HA gene. [unreadable] [unreadable] [unreadable]